Many hormones and neurotransmitters regulate the biological function via specific receptors present on cell membranes. Many of these receptors are coupled with guanine nucleotide-binding protein (hereinafter sometimes simply referred to as G protein) and mediate the intracellular signal transduction via activation of G protein, and these receptor proteins possess the common structure containing seven transmembrane domains and are thus collectively referred to as G protein-coupled receptors or seven-transmembrane receptors (7TMR).
Regulation of functions important for the living body, such as maintenance of homeostasis in the living body, reproduction, development of individuals, metabolism, growth, and regulation in the nerve system, circulatory organ system, immune system, digestive organ system and metabolic system, is carried out through interaction of hormones or neurotransmitters with G protein-coupled receptors. For regulation of biological functions, receptor proteins are known to occur for various hormones and neurotransmitters and play an important role in regulating their functions, but there still remain many unrevealed features as to unknown agonists (hormones, neurotransmitters etc.) and presence of receptors therefor.
Utilizing homology of a partial structure of G protein-coupled receptor proteins to receptor amino acid sequences, a method of searching for DNA encoding a novel receptor protein has been carried out in recent years by use of polymerase chain reaction (abbreviated hereinafter into PCR), and a large number of orphan G protein-coupled receptor proteins whose ligands are not known have been cloned (Libert, F., et al. Science, 244, 569-572, 1989, Welch, S. K., et al., Biochem. Biophys. Res. Commun., 209, 606-613, 1995, Marchese, A., et al., Genomics, 23, 609-618, 1994, Marchese, A., Genomics, 29, 335-344, 1995). Further, novel G protein-coupled receptor proteins have been found one after another by random sequencing of genomic DNA or cDNA (Nomura, N., et al., DNA Research, 1, 27-35, 1994). The general means of determining ligands for these orphan G protein-coupled receptor proteins depended conventionally on mere estimation from homology in the primary structure of the G protein-coupled receptor protein. However, a large number of orphan G protein-coupled receptor proteins have low homology to known receptors, thus making it actually difficult to estimate their corresponding ligands on only the basis of homology in the primary structure except for receptor subtypes of known ligands. On the other hand, a large number of orphan G protein-coupled receptors have been found through gene analysis, and thus it is estimated that a large number of their corresponding unknown ligands are present, but there are few reports on actual identification of ligands for orphan G protein-coupled receptors.
Recently, there are reports wherein a novel opioid peptide was searched for by introducing cDNA encoding an orphan G protein-coupled receptor protein into an animal cell (Reinsheld, R. K. et al., Science, 270, 792-794, 1995, Menular, J.-C., et al., Nature, 377, 532-535, 1995). However, the ligand in this case had been estimated to belong to a family of opioid peptides from homology to known G protein-coupled receptors and distribution in tissues. There is a long history of research and development of substances acting on the living body via opioid receptors, and various antagonists and agonists have been developed. In such development, an agonist to this receptor is found from a group of artificially synthesized compounds and used as a probe to verify expression of the receptor in cells having receptor cDNA introduced therein, and an activating substance similar to the agonist is searched for in the intracellular information transmission system and purified to determine the structure of the ligand.
Further, there is also a report wherein cDNA encoding an orphan G protein-coupled receptor (GRL104) from a snail is introduced into CHO cells, and a novel physiologically active peptide has been identified by using, as an indicator, an increase in specific intracellular calcium levels in the cells expressing the receptor (Cox, K. J. A., et al., J. Neurosci., 17(4), 1197-1205, 1997), but this novel physiologically active peptide had high homology to known leucokinin, and GRL104 was also reactive with known leucokinin. Accordingly, there are few orphan G protein-coupled receptor proteins whose ligands could be roughly estimated, and in particular when homology to the known G protein-coupled receptor protein family is low, there is no or less information on the ligand, and estimation of the ligand was difficult.
As one of reported organ G protein-coupled receptors, there is APJ (O'Dowd, B. F., et al., Gene, 436, 355-359, 1993). APJ has low homology to angiotensin receptor (AT1). A naturally occurring ligand for APJ, and its partial peptides, are described in Biochemical and Biophysical Research Communications, 251, 471-476 (1998), WO 99/33976 (Japanese Patent Application No. 220853/1998), etc., but there is no known ligand derived from a naturally occurring ligand by artificial modifications (for example, a modified ligand wherein one to several constituent amino acids in a naturally occurring ligand are replaced by other amino acids, or side chains of one to several constituent amino acids in a naturally occurring ligand are substituted with suitable substituent groups).